Direct fluorescent visualization of intracellular localization and dynamic behavior of proteins in cells and tissues in a living state is extremely important for elucidation of physiological functions of the proteins, so techniques utilizing fusion proteins comprising green fluorescent protein (GFP) have been widely used in recent years. However, a possibility of not being able to monitor target protein's behavior precisely is pointed out due to extremely large molecular size of GFP per se, a time lag between intracellular expression of GFP and formation of fluorophore, and the like.
As a method for introducing a fluorescent tag into a protein, it is known to use a thiol-reactive fluorescent labeling agent such as CPM, MDCC, fluorescein-5-maleimide and TMR-5-maleimide utilizing thiol groups in proteins (for example, thiol group of cysteine).
Although the molecular size of these thiol-reactive fluorescent labeling agents is small, they have a problem that the difference of fluorescent intensities between before and after the reaction with a thiol group is small; specifically they may emit intense fluorescence before the reaction with thiol group, or they may have very weak fluorescence after being introduced into proteins by the reaction. Moreover, since the fluorescence tag is introduced by an addition reaction with arbitrary proteins containing cysteine residues with a thiol group, they have a problem that they cannot fluorescently label the target protein with fluorescence for fluorescent-based observation.
Under these circumstances, it has been desired to provide (1) a fluorescent labeling agent having small molecular size achieving significant difference of fluorescence intensities between before and after the reaction with thiol group, and (2) a means for introducing a fluorescent tag selectively only into target protein by fluorescent labeling agent whose molecular size is small to visualize the target protein fluorescently at high sensitivity.